Treatment of intestinal epithelial cell malfunction, inflammation or damage with Hepatocyte growth factor

ABSTRACT

The present invention relates to a method and composition for treating a patient having a condition characterized as inflammatory bowel disease with an effective dose of HGF. Inflammatory bowel disease as defined by the present invention, includes Chronic Ulcerative Colitis, Crohn&#39;s Disease, necrotizing enterocolitis, severe acute gastroenteritis, chronic gastroenteritis, cholera, chronic infections of the bowel, immunologic disorders affecting the intestine, immunodeficiency syndromes affecting the intestine, and HIV. Mucosal damage and histologic lesions are reduced by administering an effective dose of HGF to patients suffering from the same. Specifically, the effective dose of HGF is in a range of about 30 μg/kg body weight/day to about 300 μg/kg body weight/day. HGF may be administered to the patient lumenally or systemically.

This application is a divisional of application Ser. No. 09/395,129,filed on Sep. 14, 1999 now U.S. Pat. No. 6,319,899 which is acontinuation-in-part of application Ser. No. 08/932,391, filed on Sep.17, 1997, now U.S. Pat. No. 5,972,887.

BACKGROUND OF THE INVENTION

The present invention relates broadly to the treatment of inflammatorybowel diseases in a patient. More particularly, the invention relates totreating a patient having an inflammatory bowel disease condition withHepatocyte Growth Factor (“HGF”).

Chronic Ulcerative Colitis (“CUC”) and Crohn's Disease (“CD”), generallyreferred to as Inflammatory Bowel Disease (“IBD”), are devastatingdisorders with an unknown etiology. Current medical therapy can controlsymptomatic exacerbations of IBD, but does not provide a cure. Progressin understanding the pathogenesis of IBD has been slowed by the lack ofavailability of animal models that exhibit the chronic, spontaneous,relapsing gastrointestinal (“GI”) inflammation that is symptomatic ofhuman IBD. Numerous murine and rat experimental models exist thatpossess some but not all of the features of human IBD.

A study has shown that the introduction of HLA-B27 and humanβ₂-microglobulin genes into Fisher (F344) rats induces spontaneouschronic GI inflammation. Hammer et al., Cell 63: 1099–1112 (1990). Inthis model, rats spontaneously develop a chronic inflammatory diseasethat includes most of the clinical and pathologic features of theB27-associated disorders in humans. The most prevalent site ofinflammation in these transgenic rats appears to be localized to thegastroinstestinal tract, and the most persistent finding is diarrheadeveloping in 100% of the animals at 20 weeks of age. Hammer et al.,Cell 63: 1099–1112 (1990); Elson et al., Gastroenterology 109: 1344–1367(1995). Because it closely approximates the human disease, as will bedescribed in detail below, this transgenic rat model was used to studythe therapeutic benefit of HGF as a treatment for IBD.

In a previous application, application Ser. No. 08/932,391, filed onSep. 17, 1997, herein specifically incorporated by reference, HGF wasshown to increase the intestinal absorptive functions and increase theintestinal tissue mass of the small intestine beyond the normal adaptiveresponse in subjects suffering from Short Bowel Syndrome, a surgicalresection of the small intestine or other developmental abnormalities ofthe small intestine. The subject of the present invention relates to thetherapeutic benefits of treating subjects with HGF who are sufferingfrom inflammation of the bowel as in IBD.

SUMMARY OF THE INVENTION

The present invention relates to a method for treating a patientcomprising administering an effective dose of HGF wherein the patienthas a condition characterized as inflammatory bowel disease.

The condition may be selected from the group consisting of ChronicUlcerative Colitis, Crohn's Disease, necrotizing enterocolitis, severeacute gastroenteritis, chronic gastroenteritis, cholera, chronicinfections of the bowel, immunologic disorders affecting the smallintestine, immunodeficiency syndromes affecting the small intestine, andHIV.

Further, the invention relates to a method for treating a patient havingintestinal mucosal damage comprising decreasing the mucosal damage ofthe small intestine by administering an effective dose of HGF to thepatient.

Still further, the invention relates to a method for treating a patienthaving histologic lesions comprising decreasing the histologic lesionsof the small intestine by administering an effective dose of HGF to thepatient.

Further, the invention includes the systemic lumenal administration ofHGF to a patient. The effective dosage range of HGF for the patient isabout 30 μg/kg body weight/day to about 300 μg/kg body weight/day.Preferably, the effective dose of HGF is about 150 μg/kg bodyweight/day.

Further, the invention relates to a composition for treating a patienthaving a condition characterized as inflammatory bowel diseasecomprising an effective dose of HGF in a suitable carrier. The suitablecarrier may be selected from the group consisting of intravenous fluidand sustained release enteral preparations.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

It has been discovered that HGF is useful for treating patientssuffering from IBD. As used herein, Inflammatory Bowel Disease or IBDincludes not only Chronic Ulcerative Colitis (“CUC”) and Crohn's Disease(“CD”) but includes necrotizing enterocolitis, severe acutegastroenteritis, chronic gastroenteritis, cholera, as well as otherchronic infections of the bowel.

Importantly, it has been discovered that administering HGF to subjectscharacterized as having IBD reduces the gross and histologic lesions inthese subjects. Further, HGF reduces the gene expression of inflammatorymediators such as TNF-α and INF-γ in these subjects.

It will be appreciated that the present invention will also haveapplication for treating intestinal disorders in subjects havingimmunologic disorders and immunodeficiency syndromes such as HIV and thelike.

HGF can be administered to patients at effective doses and for effectiveperiods of time by the intestinal intralumenal route either by catheteror sustained release preparations or by a systemic route including butnot limited to intravenous administration. Suitable carriers for HGF maybe found in Remington's Pharmaceutical Sciences, 18^(th) ed., 1990, MackPublishing Co., Easton, Pa. An effective dose of HGF is that amount ofHGF administered to a subject having an IBD condition that is sufficientto reduce gross or histologic lesions in the intestine of the subject.Preferably, the effective dose of HGF is between about 30 μg/kg bodyweight/day and about 300 μg/kg body weight/day. Most preferably, theeffective dose of HGF is about 150 μg/kg body weight/day. Subjects orpatients include, but are not limited to, rats, animals, and humans.

The following is designed merely to provide exemplification of thepreferred embodiments of the invention, and should not be construed asproviding any limitation on the scope of the invention which isdescribed in the specification and the appended claims.

Five adult Fisher 344 (F344) rats (Harlan Sprague-Dawley, Indianapolis,Ind.) and 9 adult HLA-B27 rats (Taconic Transgenic Division, Germantown,N.Y.) aged 20–24 weeks were studied. Five HLA-B27 rats underwentplacement of a jugular venous catheter connected to a subcutaneouslyplaced osmotic pump (model 2002, Alza Corp., Palo Alto, Calif.). Ratswere divided into three groups: Group 1 contained five (5) normal F344rats receiving no treatment; Group 2 contained four (4) F344-HLA-B27rats receiving no treatment; and Group 3 contained five (5) F344-HLA-B27rats receiving HGF at 150 μg/kg body weight/day. Recombinant human HGFwas provided by the Mitsubishi Chemical Corporation. After 14 days, therats were sacrificed and the gastrointestinal tract, from the Ligamentof Treitz to the rectum, was resected and opened along itsantimesenteric border. Total mucosal damage (expressed as % surface areadamaged) was measured using Image Analysis Software (Sigmascan 2.0).

With reference now to Table I, the mucosal damage, histologic lesionscores are shown for each of the three groups of rats. Group 1 consistsof F344 rats, while Group 2 consists of HLA-B27 rats receiving notreatment and Group 3 consists of HLA-B27 rats receiving HGF at 150μg/kg body weight/day. The mucosal damage and histologic lesion scoresare determined by methods well known to those skilled in the art. TheF344 rats of Group 1 did not demonstrate evidence of gross or histologiclesions in the small or large intestine. As can be seen in Table I, theadministration of HGF significantly reduced the gross (90% decrease,p<0.01) and histologic (53% decrease, p<0.01) lesions in the Group 3rats (HLA-B27+HGF) when compared to the rats in Group 2 (HLA-B27) thatdid not receive HGF.

The RNA concentration and purity were determined by measuring theabsorbency at 260 and 280 nm. One microgram of total RNA wasreverse-transcribed and the cDNA sequence was amplified using thefollowing primers: Tumor Necrosis Factor-α (TNF-α); Interferon-γ(INF-γ); Interleukin-2 (IL-2); and glyceraldehyde-3-phosphatedehydrogenase (GAPDH)— the intestinal standard. The temperature profileof the PCR reactions consisted of a 2 minute melting step at 95° C.,then 25 cycles of 30 seconds at 94° C., 30 seconds at 63° C., and 1minute at 75° C., followed by a final extension step of 5 minutes at 65°C. Independent experiments established that 25 cycles were within thelinear range of the multiplex PCR assay. RT/PCR products were separatedby size on a 4% agarose gel and stained with ethidium bromide. Imageswere transferred to an Apple Macintosh Quadra 800 via an Eagle Eye stillvideo imaging system and the relative band intensities were analyzedusing NIH image analysis software. Statistical analysis was performedusing Student's t-test and expressed as mean±SEM.

Table I shows the mean band intensities for TNF-α, INF-γ, and IL-2 foreach of the three groups of rats. The administration of HGFsignificantly reduced the gene expression of the inflammatory mediators,TNF-α (52% decrease, p<0.01) and INF-γ (93% decrease, p<0.01) in Group 3when compared to rats in Group 2. IL-2 gene expression was notdetectable in any of the groups.

The present invention demonstrates that HGF reduces the gross andhistologic intestinal lesions normally present in transgenic rats whencompared to non-treated animals. This beneficial effect is supported bya reduction in the gene expression of the inflammatory mediators TNF-αand INF-γ.

Those persons skilled in the art will therefore readily understand thatthe present invention is susceptible of a broad utility and application.Many embodiments and adaptations of the present invention other thanthose herein described, as well as many variations, modifications andequivalent arrangements, will be apparent from or reasonably suggestedby the present invention and the foregoing description thereof, withoutdeparting from the substance or scope of the present invention.Accordingly, while the present invention has been described herein indetail in relation to its preferred embodiment, it is to be understoodthat this disclosure is only illustrative and exemplary of the presentinvention and is made merely for purposes of providing a full andenabling disclosure of the invention. The foregoing disclosure is notintended or to be construed to limit the present invention or otherwiseto exclude any such other embodiments, adaptations, variations,modifications and equivalent arrangements, the present invention beinglimited only by the claims appended hereto and the equivalents thereof.

TABLE I His- Mucosal tologic Mean Band Intensity Damage Lesion TNF-α/IL-2/ Groups (%) Score GAPDH INF-γ/GAPDH GAPDH 1 0.0 0.4 ± 0.0 0.0 0.0025 2 8.5 ± 6.0 ± 0.8 ± 0.07  0.67 ± 0.12  0.0 0.96 0.41 3 0.85 ± 2.8 ±0.4 ± 0.05** 0.05 ± 0.03** 0.0 0.85** 0.5** **p < 0.01

1. A method of reducing mucosal damage in a patient having a conditioncharacterized as inflammatory bowel disease (IBD) by administering aneffective dose of HGF to the patient wherein the effective dose of HGFreduces lesions of the intestine and reduces the gene expression ofcytokine inflammatory mediators.
 2. The method of claim 1, wherein theadministration of HGF is performed systemically.
 3. The method of claim1, wherein the administration of HGF is performed lumenally.
 4. Themethod of claim 1, wherein the effective dosage of HGF is about 30 μg/kgbody weight/day to about 300 μg/kg body weight/day.
 5. The method ofclaim 1, wherein the effective dosage of HGF is about 150 μg/kg bodyweight/day.
 6. The method of claim 1, wherein the IBD is caused by oneor more conditions selected from the group consisting of ChronicUlcerative Colitis, Crohn's Disease, necrotizing enterocolitis, severeacute gastroenteritis, chronic gastroenteritis, cholera, chronicinfections of the bowel, immunologic disorders affecting the intestine,immunodeficiency syndromes affecting the intestine, and HIV.
 7. Themethod of claim 1, wherein the intestine includes the small intestine.8. The method of claim 1, wherein the cytokine inflammatory mediatorsare at least one cytokine selected from the group consisting of tumornecrosis factor-α (TNF-α) and interferon-γ (IFN-γ).
 9. The method ofclaim 1, wherein the lesions are gross lesions of the intestine.
 10. Themethod of claim 1, wherein the lesions are microscopic lesions of theintestine.